This invention relates to processes for producing immunoglobulin A in mammals, processes for producing milk containing immunoglobulin A and to the uses of the immunoglobulin A and milk produced.
Immunoglobulin A (IgA) is a well documented immunoglobulin present in almost all body fluids. It is thought to play a major role in the protection of the host from infection by pathogenic organisms invading via the mucosal surfaces of the respiratory, gastrointestinal and urogenital tracts. IgA participates in the clearance of pathogenic bacterial, viral or parasitical organisms and a variety of ingested or inhaled antigens from the mucosal surfaces by neutralising toxins and viral particles, inhibiting adherence of bacterial pathogens and preventing colonisation and penetration of mucosal surfaces by pathogenic microorganisms. The key role of immunoglobulins including IgA in milk therefore is to provide local protective immunity in the gastrointestinal tract of the offspring during the suckling period.
Immunoglobulins have come to be recognised as useful in the pharmaceutical and veterinary fields for treating bacterial or viral infections of the gut, and more generally in the treatment of disease and inflammation. Over the years various techniques for producing immunoglobulins have been proposed. A particularly popular method is for the induction and harvesting of immunoglobulins from ruminant milk. This approach has particular advantages in that the immunoglobulin produced in the milk is in a form suitable for immediate consumption, or may be processed into appropriate formulae or products. It is safe to use and the industry infrastructure for producing milk containing antibodies is already in place.
The ruminant immune system appears to differ from its human counterpart in that the immunoglobulin dominant in bovine mammary secretions is IgG1. Accordingly, the main focus of antibody production in milk by active immunisation has been on immunoglobulin G""s, although theoretically, the preferred immunoglobulin would be IgA for the reasons outlined above.
Some attempts have been made to produce increased levels of IgA in ruminant milk. Proposals for vaccination by a single administration route such as parenteral, subcutaneous, intravenous, systemic, oral, intraperitoneal, intramuscular, intramammary and the like have been suggested. Generally, these routes of administration have resulted in the predominant production of IgG1. Systemic immunisation produced both IgA and IgM in milk, but only at low concentrations. The response was enhanced when intramuscular/sub-cutaneous (IM) and intramammary (IMM) immunisation processes were combined (Am. J. Vet. Res1). Combinations of intraperitoneal (IP) and intramammary (IMM) infusion have also been shown to produce IgA and IgG, (Immunology7; Res. in Vet.Sci8, Res. in Vet.Sci11, The Ruminant Immune System in Health and Disease10. It is noted that this route leads to limited enhancement of IgA production (The Ruminant Immune System in Health and Disease10). A combination of IM and IMM immunisation gave rise to a predominance of IgG1 in the milk (Aus. J. Dairy Technology6), as well as increasing generally the levels of IgG2, IgA, and IgM (Am. J. Vet. Res1). Significant between animal variability in the antibody titres produced was also noted.
The predominance of the production of IgG1 is consistent with the findings that IgG""s produced are the major immunoglobulins in ruminant mammary secretions.
Intramammary immunisation techniques have generally not been preferred as a route for vaccination under field conditions due to the high chance of mammary infection (Aus. J. Dairy Technology6). However, other work suggests that this may not be the case (Am. J. Vet. Res1).
It should be noted that much of the published literature concerning immunoglobulin production in mammary gland secretions is directed to disease prevention in animals or their offspring. Few are directed to the production of immunoglobulin enriched milk for the purposes of obtaining the immunoglobulins themselves.
An exception to this is a process for the production of a protein concentrate containing immunological factors of lactic origin in Swiss Patent No. 1,573,995. Nearly 20 years ago, this patent disclosed a process for producing milk with a high antibody titre, by intracisternal instillation into the mammary gland, parental injection (subcutaneous, intravenous), injection into the retromammary ganglionic system by scarification, by oral ingestion or by a combination of several of these modes. The only specific immunisation protocol for obtaining colostral and transition milk disclosed involved some 11 immunisation steps over a period of 8 weeks prior to calving. This protocol comprises multiple parental (including intravenous) administration steps, with several IMM administration steps interspersed and requires 2 oral administration steps in the week prior to calving.
This protocol is not in widespread use today. The immunisation plan is onerous in the number of steps involved and is not in fact optimised for immunoglobulin A production. Indeed, the patent is misleading in suggesting that IgA""s are preponderant in ruminant maternal milk; a misconception that may have resulted from the knowledge that IgA is predominant in human milk. As established in other teachings (see for example, Aus. J. Dairy Technology6), IgG is the predominant immunoglobulin produced in the maternal milk.
It has also been shown in the intervening years that oral delivery of antigens results in little or no increase of IgA titres in mammary secretions when compared with non-inoculated controls (Am. J. Vet. R1). It is presumed that the presence of the rumen may preclude the antigen reaching the small intestine. Accordingly, the oral administration step called for by Hilpert is now contraindicated.
Similarly, intravenous injection would not generally be recommended for immunisation purposes because of the possible adverse effects such as anaphylactic shock (Cold Spring Harbour18, ILAR Journal19).
There is currently a need for a process for inducing, and producing IgA in milk at higher levels than have previously been obtained by known antigen administration processes. A process which additionally reduces between animal variability in the production of IgA is also desirable. A commercial process which optimises production of IgA while simplifying the immunisation protocol is also sought.
It is therefore an object of this invention to provide a process for the induction and production of immunoglobulin A in milk which goes some way towards overcoming the above disadvantages or at least provides the public with a useful choice.
Accordingly, the present invention can broadly be said to consist in a process for the induction of immunoglobulin A (IgA) in a mammal which process comprises:
(a) actively immunising a pregnant mammal with an antigen by any two routes of administration selected from intramammary (IMM), intraperitoneal (IP) and intramuscular (IM); and
(b) actively immunising said mammal with an antigen by a third administration route selected from intramammary (IMM), intraperitoneal (IP) and intramuscular (IM); with the proviso that all three administration routes are different.
In a further aspect the present invention provides a process for the production of mammalian milk containing immunoglobulin A (IgA), which process comprises:
(a) induction of IgA according to the process set out above; and
(b) collecting milk containing IgA from said mammal.
Preferably, the initial immunisation protocol is followed by a programme of booster immunisations over the preparturition period.
In a preferred process of the present invention the antigen administered is the same for each route of administration.
Preferably, the antigen administered is emulsified in an adjuvant. A particularly preferred adjuvant is Freunds incomplete adjuvant (FIC).
In one embodiment of the invention IgA may be isolated from the mammalian milk collected. The isolated IgA may be purified if desired.
In a further aspect, the present invention provides mammalian milk containing IgA produced in accordance with the processes of the invention.
In a still further aspect, the present invention provides IgA produced in accordance with the processes of the invention.
Preferred mammals for use in the processes of the present invention are ruminants, especially diary cows.
The present invention further provides for the use of immunoglobulin A produced in accordance with the processes of the invention in pharmaceutical, cosmetic and veterinary compositions as well as in food products including functional foods and dietary supplements.